Fusion proteins of mycobacterium tuberculosis antigens and their uses

ABSTRACT

The present invention relates to fusion proteins of  Mycobacterium tuberclosis  antigens. In particular, it relates to two fusion proteins, each of which contains three individual  M. tuberculosis  antigens, and a fusion protein of two  M. tuberculosis  antigens, their coding sequences, and methods for their use in the treatment and prevention of tuberculosis.

2. BACKGROUND OF THE INVENTION

Tuberculosis is a chronic infectious disease caused by infection with M.tuberculosis. It is a major disease in developing countries, as well asan increasing problem in developed areas of the world, with about 8million new cases and 3 million deaths each year. Although the infectionmay be asymptomatic for a considerable period of time, the disease ismost commonly manifested as an acute inflammation of the lungs,resulting in fever and a nonproductive cough. If untreated, seriouscomplications and death typically result.

Although tuberculosis can generally be controlled using extendedantibiotic therapy, such treatment is not sufficient to prevent thespread of the disease. Infected individuals may be asymptomatic, butcontagious, for some time. In addition, although compliance with thetreatment regimen is critical, patient behavior is difficult to monitor.Some patients do not complete the course of treatment, which can lead toineffective treatment and the development of drug resistance.

In order to control the spread of tuberculosis, effective vaccination,and accurate early diagnosis of the disease are of utmost importance.Currently, vaccination with live bacteria is the most efficient methodfor inducing protective immunity. The most common Mycobacterium employedfor this purpose is Bacillus Calmette-Guerin (BCG), an avirulent strainof M. bovis. However, the safety and efficacy of BCG is a source ofcontroversy and some countries, such as the United States, do notvaccinate the general public with this agent.

Diagnosis of tuberculosis is commonly achieved using a skin test, whichinvolves intradermal exposure to tuberculin PPD (protein-purifiedderivative). Antigen-specific T cell responses result in measurableinduration at the injection site by 48-72 hours after injection, whichindicates exposure to Mycobacterial antigens. Sensitivity andspecificity have, however, been a problem with this test, andindividuals vaccinated with BCG cannot be distinguished from infectedindividuals.

While macrophages have been shown to act as the principal effectors ofM. tuberculosis immunity, T cells are the predominant inducers of suchimmunity. The essential role of T cells in protection against M.tuberculosis infection is illustrated by the frequent occurrence of M.tuberculosis in Acquired Immunodeficiency Syndrome patients, due to thedepletion of CD4⁺ T cells associated with human immunodeficiency virus(HIV) infection. Mycobacterium-reactive CD4⁺ T cells have been shown tobe potent producers of gamma-interferon (IFN-γ), which, in turn, hasbeen shown to trigger the anti-mycobacterial effects of macrophages inmice. While the role of IFN-γ in humans is less clear, studies haveshown that 1,25-dihydroxy-vitamin D3, either alone or in combinationwith IFN-γ or tumor necrosis factor-alpha, activates human macrophagesto inhibit M. tuberculosis infection. Furthermore, it is known thatIFN-γ stimulates human macrophages to make 1,25-dihydroxy-vitamin D3.Similarly, interleukin-12 (IL-12) has been shown to play a role instimulating resistance to M. tuberculosis infection. For a review of theimmunology of M. tuberculosis infection, see Chan and Kaufmann, 1994,Tuberculosis: Pathogenesis, Protection and Control, Bloom (ed.), ASMPress, Washington, DC.

Accordingly, there is a need for improved vaccines, and methods forpreventing and treating tuberculosis.

3. SUMMARY OF THE INVENTION

The present invention relates to fusion proteins of M. tuberculosisantigens. In particular, it relates to fusion polypeptides that containtwo or three M. tuberculosis antigens, polynucleotides encoding suchpolypeptides, methods of using the polypeptides and polynucleotides inthe treatment and prevention of M. tuberculosis infection.

The present invention is based, in part, on Applicants' discovery thattwo polynucleotides, each containing three M. tuberculosis codingsequences, produced recombinant fusion proteins that retained theimmunogenicity and antigenicity of their individual components. Thefusion proteins induced both T cell and B cell responses, as measured byT cell proliferation, cytokine production, and antibody production.Furthermore, a fusion protein was used as an immunogen with adjuvants invivo to elicit both cell-mediated and humoral immunity to M.tuberculosis. Additionally, a fusion protein of two antigens was made bya fusion construct and used in a vaccine formulation with an adjuvant toafford long-term protection in animals against the development oftuberculosis. The fusion protein was a more effective immunogen than amixture of the two proteins.

In a specific embodiment of the invention, the isolated or purified M.tuberculosis polypeptides of the invention may be formulated aspharmaceutical compositions for administration into a subject in theprevention and/or treatment of M. tuberculosis infection. Theimmunogenicity of the fusion protein may be enhanced by the inclusion ofan adjuvant.

In another aspect of the invention, the isolated or purifiedpolynucleotides are used to produce recombinant fusion polypeptideantigens in vitro. Alternatively, the polynucleotides may beadministered directly into a subject as DNA vaccines to cause antigenexpression in the subject, and the subsequent induction of an anti-M.tuberculosis immune response.

4. BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B. The nucleotide sequence (SEQ ID NO:1) and amino acidsequence (SEQ ID NO:2) of tri-fusion protein Ra12-TbH9-Ra35.

FIG. 2: The nucleotide sequence (SEQ ID NO:3) and amino acid sequence(SEQ ID NO:4) of tri-fusion protein Erd14-DPV-MT1.

FIGS. 3A-3F: T cell proliferation responses of six PPD+ subjects whenstimulated with two fusion proteins and their individual components.

FIGS. 4A-4F: IFN-γ production of six PPD+ subjects when stimulated withtwo fusion proteins and their individual components.

FIGS. 5A-5F: T cell proliferation of mice immunized with a fusionprotein or its individual components and an adjuvant.

FIG. 6: IFN-γ production of mice immunized with a fusion protein or itsindividual components and an adjuvant.

FIG. 7: IL-4 production of mice immunized with a fusion protein or itsindividual components and an adjuvant.

FIGS. 8A-8F: Serum antibody concentrations of mice immunized with afusion protein or its individual components and an adjuvant.

FIGS. 9A-9C: Survival of guinea pigs after aerosol challenge of M.tuberculosis. Mtb32A and Mtb39A were formulated in adjuvant SBAS1c (9A),SBAS2 (9B) or SBAS7 (9C), and used as an immunogen in guinea pigs priorto challenge with bacteria. BCG is the positive control.

5. DETAILED DESCRIPTION OF THE INVENTION

5.1. Isolation of Coding Sequences

The present invention relates to nucleic acid molecules that encodefusion polypeptides of M. tuberculosis. In a specific embodiment by wayof example in Section 6, infra, three M. tuberculosis fusion codingsequences were constructed and expressed. In accordance with theinvention, any nucleotide sequence which encodes the amino acid sequenceof the fusion protein can be used to generate recombinant moleculeswhich direct the expression of the coding sequence.

In order to clone full-length coding sequences or homologous variants togenerate the fusion polynucleotides, labeled DNA probes designed fromany portion of the nucleotide sequences or their complements disclosedherein may be used to screen a genomic or cDNA library made from variousstrains of M. tuberculosis to identify the coding sequence of eachindividual component. Isolation of coding sequences may also be carriedout by the polymerase chain reactions (PCR) using two degenerateoligonucleotide primer pools designed on the basis of the codingsequences disclosed herein.

The invention also relates to isolated or purified polynucleotidescomplementary to the nucleotide sequences of SEQ ID NOS: 1 and 3, andpolynucleotides that selectively hybridize to such complementarysequences. In a preferred embodiment, a polynucleotide which hybridizesto the sequence of SEQ ID NOS: 1 and 3 or its complementary sequenceunder conditions of low stringency and encodes a protein that retainsthe immunogenicity of the fusion proteins of SEQ ID NOS:2 and 4 isprovided. By way of example and not limitation, exemplary conditions oflow stringency are as follows (see also Shilo and Weinberg, 1981, Proc.Natl. Acad. Sci. USA 78:6789-6792): Filters containing DNA arepretreated for 6 h at 40° C. in absolution containing 35% formamide,5×SSC, 50 mM Tris-HCl (pH 7.5), 5 mM EDTA, 0.1% PVP, 0.1% Ficoll, 1%BSA, and 500 μg/ml denatured salmon sperm DNA. Hybridizations arecarried out in the same solution with the following modifications: 0.02%PVP, 0.02% Ficoll, 0.2% BSA, 100 μg/ml salmon sperm DNA, 10% (wt/vol)dextran sulfate, and 5-20×10⁶ cpm ³²P-labeled probe is used. Filters areincubated in hybridization mixture for 18-20 h at 40° C., and thenwashed for 1.5 h at 55° C. in a solution containing 2×SSC, 25 mMTris-HCl (pH 7.4), 5 mM EDTA, and 0.1% SDS. The wash solution isreplaced with fresh solution and incubated an additional 1.5 h at 60° C.Filters are blotted dry and exposed for autoradiography. If necessary,filters are washed for a third time at 65-68° C. and re-exposed to film.Other conditions of low stringency which may be used are well known inthe art (e.g., as employed for cross-species hybridizations).

In another preferred embodiment, a polynucleotide which hybridizes tothe coding sequence of SEQ ID NOS:1 and 3 or its complementary sequenceunder conditions of high stringency and encodes a protein that retainsthe immunogenicity of the fusion proteins of SEQ ID NOS:2 and 4 isprovided. By way of example and not limitation, exemplary conditions ofhigh stringency are as follows: Prehybridization of filters containingDNA is carried out for 8 h to overnight at 65° C. in buffer composed of6×SSC, 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.02% PVP, 0.02% Ficoll,0.02% BSA, and 500 μg/ml denatured salmon sperm DNA. Filters arehybridized for 48 h at 65° C. in prehybridization mixture containing 100μg/ml denatured salmon sperm DNA and 5-20×10⁶ cpm of ³²P-labeled probe.Washing of filters is done at 37° C. for 1 h in a solution containing2×SSC, 0.01% PVP, 0.01% Ficoll, and 0.01% BSA. This is followed by awash in 0.1×SSC at 50° C. for 45 min before autoradiography. Otherconditions of high stringency which may be used are well known in theart.

In yet another preferred embodiment, a polynucleotide which hybridizesto the coding sequence of SEQ ID NOS:1 and 3 or its complementarysequence under conditions of moderate stringency and encodes a proteinthat retains the immunogenicity of the fusion proteins of SEQ ID NOS:2and 4 is provided. Exemplary conditions of moderate stringency are asfollows: Filters containing DNA are pretreated for 6 h at 55° C. in asolution containing 6×SSC, 5×Denhart's solution, 0.5% SDS and 100 μg/mldenatured salmon sperm DNA. Hybridizations are carried out in the samesolution and 5-20×10⁶ cpm ³²P-labeled probe is used. Filters areincubated in hybridization mixture for 18-20 h at 55° C., and thenwashed twice for 30 minutes at 60° C. in a solution containing 1×SSC and0.1% SDS. Filters are blotted dry and exposed for autoradiography. Otherconditions of moderate stringency which may be used are well-known inthe art. Washing of filters is done at 37° C. for 1 h in a solutioncontaining 2×SSC, 0.1% SDS.

5.2. Polypeptides Encoded by the Coding Sequences

In accordance with the invention, a polynucleotide of the inventionwhich encodes a fusion protein, fragments thereof, or functionalequivalents thereof may be used to generate recombinant nucleic acidmolecules that direct the expression of the fusion protein, fragmentsthereof, or functional equivalents thereof, in appropriate host cells.The fusion polypeptide products encoded by such polynucleotides may benaturally occurring or altered by molecular manipulation of the codingsequence.

Due to the inherent degeneracy of the genetic code, other DNA sequenceswhich encode substantially the same or a functionally equivalent aminoacid sequence, may be used in the practice of the invention for theexpression of the fusion polypeptides. Such DNA sequences include thosewhich are capable of hybridizing to the coding sequences or theircomplements disclosed herein under low, moderate or high stringencyconditions described in Sections 5.1, supra.

Altered nucleotide sequences which may be used in accordance with theinvention include deletions, additions or substitutions of differentnucleotide residues resulting in a sequence that encodes the same or afunctionally equivalent gene product. The gene product itself maycontain deletions, additions or substitutions of amino acid residues,which result in a silent change thus producing a functionally equivalentantigenic epitope. Such conservative amino acid substitutions may bemade on the basis of similarity in polarity, charge, solubility,hydrophobicity, hydrophilicity, and/or the amphipathic nature of theresidues involved. For example, negatively charged amino acids includeaspartic acid and glutamic acid; positively charged amino acids includelysine, histidine and arginine; amino acids with uncharged polar headgroups having similar hydrophilicity values include the following:glycine, asparagine, glutamine, serine, threonine and tyrosine; andamino acids with nonpolar head groups include alanine, valine,isoleucine, leucine, phenylalanine, proline, methionine and tryptophan.

The nucleotide sequences of the invention may be engineered in order toalter the fusion protein coding sequence for a variety of ends,including but not limited to, alterations which modify processing andexpression of the gene product. For example, mutations may be introducedusing techniques which are well known in the art, e.g., site-directedmutagenesis, to insert new restriction sites, to alter glycosylationpatterns, phosphorylation, etc.

In an alternate embodiment of the invention, the coding sequence of afusion protein could be synthesized in whole or in part, using chemicalmethods well known in the art. See, e.g., Caruthers et al., 1980, Nuc.Acids Res. Symp. Ser. 7:215-233; Crea and Horn, 180, Nuc. Acids Res.9(10):2331; Matteucci and Caruthers, 1980, Tetrahedron Letter 21:719;and Chow and Kempe, 1981, Nuc. Acids Res. 9(12):2807-2817.Alternatively, the polypeptide itself could be produced using chemicalmethods to synthesize an amino acid sequence in whole or in part. Forexample, peptides can be synthesized by solid phase techniques, cleavedfrom the resin, and purified by preparative high performance liquidchromatography. (See Creighton, 1983, Proteins Structures And MolecularPrinciples, W.H. Freeman and Co., N.Y. pp. 50-60). The composition ofthe synthetic polypeptides may be confirmed by amino acid analysis orsequencing (e.g., the Edman degradation procedure; see Creighton, 1983,Proteins, Structures and Molecular Principles, W. H. Freeman and Co.,N.Y., pp. 34-49).

Additionally, the coding sequence of a fusion protein can be mutated invitro or in vivo, to create and/or destroy translation, initiation,and/or termination sequences, or to create variations in coding regionsand/or form new restriction endonuclease sites or destroy preexistingones, to facilitate further in vitro modification. Any technique formutagenesis known in the art can be used, including but not limited to,chemical mutagenesis, in vitro site-directed mutagenesis (Hutchinson,C., et al., 1978, J. Biol. Chem 253:6551), use of TAB® linkers(Pharmacia), and the like. It is important that the manipulations do notdestroy immunogenicity of the fusion polypeptides.

In addition, nonclassical amino acids or chemical amino acid analogs canbe introduced as a substitution or addition into the sequence.Non-classical amino acids include, but are not limited to, the D-isomersof the common amino acids, α-amino isobutyric acid, 4-aminobutyric acid,Abu, 2-amino butyric acid, γ-Abu, ε-Ahx, 6-amino hexanoic acid, Aib,2-amino isobutyric acid, 3-amino propionic acid, ornithine, norleucine,norvaline, hydroxyproline, sarcosine, citrulline, cysteic acid,t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine,β-alanine, fluoro-amino acids, designer amino acids such as β-methylamino acids, Cα-methyl amino acids, Nα-methyl amino acids, and aminoacid analogs in general. Furthermore, the amino acid can be D(dextrorotary) or L (levorotary).

In a specific embodiment, the coding sequences of each antigen in thefusion protein are joined at their amino- or carboxy-terminus via apeptide bond in any order. Alternatively, the antigens are connected bya flexible polylinker such as Gly-Cys-Gly or Gly-Gly-Gly-Gly-Serrepeated 1 to 3 times (SEQ ID NOS:5-7 and 8-10, respectively) (Bird etal., 1988, Science 242: 423-426; Chaudhary et al., 1990, Proc. Nat'l.Acad. Sci. U.S.A. 87:1066-1070). In one embodiment, such a protein isproduced by recombinant expression of a nucleic acid encoding theprotein. Such a fusion product can be made by ligating the appropriatenucleic acid sequences encoding the desired amino acid sequences to eachother by methods known in the art, in the proper coding frame, andexpressing the product by methods commonly known in the art.Alternatively, such a product may be made by protein synthetictechniques, e.g., by use of a peptide synthesizer. Coding sequences forother molecules such as a cytokine or an adjuvant can be added to thefusion polynucleotide as well.

5.3. Production of Fusion Proteins

In order to produce a M. tuberculosis fusion protein of the invention,the nucleotide sequence coding for the protein, or a functionalequivalent, is inserted into an appropriate expression vector, i e., avector which contains the necessary elements for the transcription andtranslation of the inserted coding sequence. The host cells or celllines transfected or transformed with recombinant expression vectors canbe used for a variety of purposes. These include, but are not limitedto, large scale production of the fusion protein.

Methods which are well known to those skilled in the art can be used toconstruct expression vectors containing a fusion coding sequence andappropriate transcriptional/translational control signals. These methodsinclude in vitro recombinant DNA techniques, synthetic techniques and invivo recombination/genetic recombination. (See, e.g., the techniquesdescribed in Sambrook et al., 1989, Molecular Cloning A LaboratoryManual, Cold Spring Harbor Laboratory, N.Y. and Ausubel et al., 1989,Current Protocols in Molecular Biology, Greene Publishing Associates andWiley Interscience, N.Y.). RNA capable of encoding a polypeptide mayalso be chemically synthesized (Gait, ed., 1984, OligonucleoideSynthesis, IRL Press, Oxford).

5.3.1. Expression Systems

A variety of host-expression vector systems may be utilized to express afusion protein coding sequence. These include, but are not limited to,microorganisms such as bacteria (e.g. E. coli, B. subtilis) transformedwith recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expressionvectors containing a coding sequence; yeast (e.g. Saccharomycdes,Pichia) transformed with recombinant yeast expression vectors containinga coding sequence; insect cell systems infected with recombinant virusexpression vectors (em, baculovirus) containing a coding sequence; plantcell systems infected with recombinant virus expression vectors (es,cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) ortransformed with recombinant plasmid expression vectors (es, Ti plasmid)containing a coding sequence; or mammalian cell systems (e.g. COS, CHO,BHK, 293, 3T3 cells). The expression elements of these systems vary intheir strength and specificities.

Depending on the host/vector system utilized, any of a number ofsuitable transcription and translation elements, including constitutiveand inducible promoters, may be used in the expression vector. Forexample, when cloning in bacterial systems, inducible promoters such aspL of bacteriophage λ, plac, ptrp, ptac (ptrp-lac hybrid promoter;cytomegalovirus promoter) and the like may be used; when cloning ininsect cell systems, promoters such as the baculovirus polyhedronpromoter may be used; when cloning in plant cell systems, promotersderived from the genome of plant cells (ev, heat shock promoters; thepromoter for the small subunit of RUBISCO; the promoter for thechlorophyll α/β binding protein) or from plant viruses (ep, the 35S RNApromoter of CaMV; the coat protein promoter of TMV) may be used; whencloning in mammalian cell systems, promoters derived from the genome ofmammalian cells (es, metallothionein promoter) or from mammalian viruses(ev, the adenovirus late promoter; the vaccinia virus 7.5K promoter) maybe used; when generating cell lines that contain multiple copies of athe antigen coding sequence, SV40-, BPV- and EBV-based vectors may beused with an appropriate selectable marker.

Bacterial systems are preferred for the expression of M. tuberculosisantigens. For in vivo delivery, a bacterium such asBacillus-Calmette-Guerrin may be engineered to express a fusionpolypeptide of the invention on its cell surface. A number of otherbacterial expression vectors may be advantageously selected dependingupon the use intended for the expressed products. For example, whenlarge quantities of the fusion protein are to be produced forformulation of pharmaceutical compositions, vectors which direct theexpression of high levels of fusion protein products that are readilypurified may be desirable. Such vectors include, but are not limited to,the E. coli expression vector pUR278 (Ruther et al., 1983, EMBO J.2:1791), in which a coding sequence may be ligated into the vector inframe with the lacZ coding region so that a hybrid protein is produced;pIN vectors (Inouye & Inouye, 1985, Nucleic acids Res. 13:3101-3109; VanHeeke & Schuster, 1989, J. Biol. Chem. 264:5503-5509); and the like.pGEX vectors may also be used to express foreign polypeptides as fusionproteins with glutathione S-transferase (GST). In general, such fusionproteins are soluble and can be purified easily from lysed cells byadsorption to glutathione-agarose beads followed by elution in thepresence of free glutathione. The pGEX vectors are designed to includethrombin or factor Xa protease cleavage sites so that the cloned fusionpolypeptide of interest can be released from the GST moiety.

5.3.2. Protein Purification

Once a recombinant protein is expressed, it can be identified by assaysbased on the physical or functional properties of the product, includingradioactive labeling of the product followed by analysis by gelelectrophoresis, radioimmunoassay, ELISA, bioassays, etc.

Once the encoded protein is identified, it may be isolated and purifiedby standard methods including chromatography (e.g., high performanceliquid chromatography, ion exchange, affinity, and sizing columnchromatography), centrifugation, differential solubility, or by anyother standard technique for the purification of proteins. The actualconditions used will depend, in part, on factors such as net charge,hydrophobicity, hydrophilicity, etc., and will be apparent to thosehaving skill in the art. The functional properties may be evaluatedusing any suitable assay such as antibody binding, induction of T cellproliferation, stimulation of cytokine production such as IL2, IL-4 andIFN-γ. For the practice of the present invention, it is preferred thateach fusion protein is at least 80% purified from other proteins. It ismore preferred that they are at least 90% purified. For in vivoadministration, it is preferred that the proteins are greater than 95%purified.

5.4. Uses of the Fusion Protein Coding Sequence

The fusion protein coding sequence of the invention may be used toencode a protein product for use as an immunogen to induce and/orenhance immune responses to M. tuberculosis. In addition, such codingsequence may be ligated with a coding sequence of another molecule suchas cytokine or an adjuvant. Such polynucleotides may be used in vivo asa DNA vaccine (U.S. Pat. Nos. 5,589,466; 5,679,647; 5,703,055). In thisembodiment of the invention, the polynucleotide expresses its encodedprotein in a recipient to directly induce an immune response. Thepolynucleotide may be injected into a naive subject to prime an immuneresponse to its encoded product, or administered to an infected orimmunized subject to enhance the secondary immune responses.

In a preferred embodiment, a therapeutic composition comprises a fusionprotein coding sequence or fragments thereof that is part of anexpression vector. In particular, such a polynucleotide contains apromoter operably linked to the coding region, said promoter beinginducible or constitutive, and, optionally, tissue-specific. In anotherembodiment, a polynucleotide contains a coding sequence flanked byregions that promote homologous recombination at a desired site in thegenome, thus providing for intrachromosomal expression of the codingsequence (Koller and Smithies, 1989, Proc. Natl. Acad. Sci. USA86:8932-8935; Zijlstra et al., 1989, Nature 342:435-438).

Delivery of the nucleic acid into a subject may be either direct, inwhich case the subject is directly exposed to the nucleic acid ornucleic acid-carrying vector, or indirect, in which case, cells arefirst transformed with the nucleic acid in vitro, then transplanted intothe subject. These two approaches are known, respectively, as in vivo orex vivo gene transfer.

In a specific embodiment, the nucleic acid is directly administered invivo, where it is expressed to produce the encoded fusion proteinproduct. This can be accomplished by any of numerous methods known inthe art, e.g., by constructing it as part of an appropriate nucleic acidexpression vector and administering it so that it becomes intracellular,e.g., by infection using a defective or attenuated retroviral or otherviral vector (see U.S. Pat. No. 4,980,286), or by direct injection ofnaked DNA, or by use of microparticle bombardment (e.g., a gene gun;Biolistic, Dupont), or coating with lipids or cell-surface receptors ortransfecting agents, encapsulation in liposomes, microparticles, ormicrocapsules, or by administering it in linkage to a peptide which isknown to enter the nucleus, by administering it in linkage to a ligandsubject to receptor-mediated endocytosis (see e.g., Wu and Wu, 1987, J.Biol. Chem. 262:4429-4432) which can be used to target cell typesspecifically expressing the receptors, etc. In another embodiment, anucleic acid-ligand complex can be formed in which the ligand comprisesa fusogenic viral peptide to disrupt endosomes, allowing the nucleicacid to avoid lysosomal degradation. In yet another embodiment, thenucleic acid can be targeted in vivo for cell specific uptake andexpression, by targeting a specific receptor (see, e.g., PCTPublications WO 92/06180 dated Apr. 16, 1992; WO 92/22635 dated Dec. 23,1992; WO92/20316 dated Nov. 26, 1992; WO93/14188 dated Jul. 22, 1993; WO93/20221 dated Oct. 14, 1993). Alternatively, the nucleic acid can beintroduced intracellularly and incorporated within host cell DNA forexpression, by homologous recombination (Koller and Smithies, 1989,Proc. Natl. Acad. Sci. USA 86:8932-8935; Zijlstra et al., 1989, Nature342:435-438).

In a specific embodiment, a viral vector such as a retroviral vector canbe used (see Miller et al., 1993, Meth. Enzymol. 217:581-599).Retroviral vectors have been modified to delete retroviral sequencesthat are not necessary for packaging of the viral genome and integrationinto host cell DNA. A fusion coding sequence is cloned into the vector,which facilitates delivery of the nucleic acid into a recipient. Moredetail about retroviral vectors can be found in Boesen et al., 1994,Biotherapy 6:291-302, which describes the use of a retroviral vector todeliver the mdrl gene to hematopoietic stem cells in order to make thestem cells more resistant to chemotherapy. Other references illustratingthe use of retroviral vectors in gene therapy are: Clowes et al., 1994,J. Clin. Invest. 93:644-651; Kiem et al., 1994, Blood 83:1467-1473;Salmons and Gunzberg, 1993, Human Gene Therapy 4:129-141; and Grossmanand Wilson, 1993, Curr. Opin. in Genetics and Devel. 3:110-114.

Adenoviruses are other viral vectors that can be used in gene therapy.Adenoviruses are especially attractive vehicles for delivering genes torespiratory epithelia. Adenoviruses naturally infect respiratoryepithelia where they cause a mild disease. Other targets foradenovirus-based delivery systems are liver, the central nervous system,endothelial cells, and muscle. Adenoviruses have the advantage of beingcapable of infecting non-dividing cells. Adeno-associated virus (AAV)has also been proposed for use in in vivo gene transfer (Walsh et al.,1993, Proc. Soc. Exp. Biol. Med. 204:289-300.

Another approach involves transferring a construct to cells in tissueculture by such methods as electroporation, lipofection, calciumphosphate mediated transfection, or viral infection. Usually, the methodof transfer includes the transfer of a selectable marker to the cells.The cells are then placed under selection to isolate those cells thathave taken up and are expressing the transferred gene. Those cells arethen delivered to a subject.

In this embodiment, the nucleic acid is introduced into a cell prior toadministration in vivo of the resulting recombinant cell. Suchintroduction can be carried out by any method known in the art,including but not limited to transfection, electroporation,microinjection, infection with a viral or bacteriophage vectorcontaining the nucleic acid sequences, cell fusion, chromosome-mediatedgene transfer, microcell-mediated gene transfer, spheroplast fusion,etc. Numerous techniques are known in the art for the introduction offoreign genes into cells (see e.g., Loeffler and Behr, 1993, Meth.Enzymol. 217:599-618; Cohen et al., 1993, Meth. Enzymol. 217:618-644;Cline, 1985, Pharmac. Ther. 29:69-92) and may be used in accordance withthe present invention.

5.5. Uses of the Fusion Protein

Purified or partially purified fusion proteins or fragments thereof maybe formulated as a vaccine or therapeutic composition. Such compositionmay include adjuvants to enhance immune responses. Commonly usedadjuvants include, but are not limited to, aluminum hydroxide, mineraloil, lipid A and B. pertussis. In addition, such proteins may be furthersuspended in an oil emulsion to cause a slower release of the proteinsin vivo upon injection. The optimal ratios of each component in theformulation may be determined by techniques well known to those skilledin the art.

Such a formulation may be administered to a subject per se or in theform of a pharmaceutical or therapeutic composition. Pharmaceuticalcompositions comprising the proteins may be manufactured by means ofconventional mixing, dissolving, granulating, dragee-making, levigating,emulsifying, encapsulating, entrapping or lyophilizing processes.Pharmaceutical compositions may be formulated in conventional mannerusing one or more physiologically acceptable carriers, diluents,excipients or auxiliaries which facilitate processing of thepolypeptides into preparations which can be used pharmaceutically.Proper formulation is dependent upon the route of administration chosen.

For topical administration, the proteins may be formulated as solutions,gels, ointments, creams, suspensions, etc. as are well-known in the art.

Systemic formulations include those designed for administration byinjection, e.g. subcutaneous, intravenous, intramuscular, intrathecal orintraperitoneal injection, as well as those designed for transdermal,transmucosal, oral or pulmonary administration.

For injection, the proteins may be formulated in aqueous solutions,preferably in physiologically compatible buffers such as Hanks'ssolution, Ringer's solution, or physiological saline buffer. Thesolution may contain formulatory agents such as suspending, stabilizingand/or dispersing agents. Alternatively, the proteins may be in powderform for constitution with a suitable vehicle, e.g., sterilepyrogen-free water, before use.

For transmucosal administration, penetrants appropriate to the barrierto be permeated are used in the formulation. Such penetrants aregenerally known in the art.

For oral administration, a composition can be readily formulated bycombining the proteins with pharmaceutically acceptable carriers wellknown in the art. Such carriers enable the proteins to be formulated astablets, pills, dragees, capsules, liquids, gels, syrups, slurries,suspensions and the like, for oral ingestion by a subject to be treated.For oral solid formulations such as, for example, powders, capsules andtablets, suitable excipients include fillers such as sugars, such aslactose, sucrose, mannitol and sorbitol; cellulose preparations such asmaize starch, wheat starch, rice starch, potato starch, gelatin, gumtragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodiumcarboxymethylcellulose, and/or polyvinylpyrrolidone (PVP); granulatingagents; and binding agents. If desired, disintegrating agents may beadded, such as the cross-linked polyvinylpyrrolidone, agar, or alginicacid or a salt thereof such as sodium alginate.

If desired, solid dosage forms may be sugar-coated or enteric-coatedusing standard techniques.

For oral liquid preparations such as, for example, suspensions, elixirsand solutions, suitable carriers, excipients or diluents include water,glycols, oils, alcohols, etc. Additionally, flavoring agents,preservatives, coloring agents and the like may be added.

For buccal administration, the proteins may take the form of tablets,lozenges, etc. formulated in conventional manner.

For administration by inhalation, the proteins for use according to thepresent invention are conveniently delivered in the form of an aerosolspray from pressurized packs or a nebulizer, with the use of a suitablepropellant, e.g., dichlorodifluoromethane, trichlorofluoromethane,dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In thecase of a pressurized aerosol the dosage unit may be determined byproviding a valve to deliver a metered amount. Capsules and cartridgesof e.g. gelatin for use in an inhaler or insufflator may be formulatedcontaining a powder mix of the proteins and a suitable powder base suchas lactose or starch.

The proteins may also be formulated in rectal or vaginal compositionssuch as suppositories or retention enemas, e.g, containing conventionalsuppository bases such as cocoa butter or other glycerides.

In addition to the formulations described previously, the proteins mayalso be formulated as a depot preparation. Such long acting formulationsmay be administered by implantation (for example subcutaneously orintramuscularly) or by intramuscular injection. Thus, for example, theproteins may be formulated with suitable polymeric or hydrophobicmaterials (for example as an emulsion in an acceptable oil) or ionexchange resins, or as sparingly soluble derivatives, for example, as asparingly soluble salt.

Alternatively, other pharmaceutical delivery systems may be employed.Liposomes and emulsions are well known examples of delivery vehiclesthat may be used to deliver an antigen. Certain organic solvents such asdimethylsulfoxide also may be employed, although usually at the cost ofgreater toxicity. Additionally, the proteins may be delivered using asustained-release system, such as semipermeable matrices of solidpolymers containing the therapeutic or vaccinating agent. Varioussustained-release materials have been established and are well known bythose skilled in the art. Sustained-release capsules may, depending ontheir chemical nature, release the proteins for a few weeks up to over100 days. Depending on the chemical nature and the biological stabilityof the reagent, additional strategies for protein stabilization may beemployed.

Determination of an effective amount of the fusion protein for inducingan immune response in a subject is well within the capabilities of thoseskilled in the art, especially in light of the detailed disclosureprovided herein.

An effective dose can be estimated initially from in vitro assays. Forexample, a dose can be formulated in animal models to achieve aninduction of an immune response using techniques that are well known inthe art. One having ordinary skill in the art could readily optimizeadministration to humans based on animal data. Dosage amount andinterval may be adjusted individually. For example, when used as avaccine, the polypeptides and/or polynucleotides of the invention may beadministered in about 1 to 3 doses for a 1-36 week period. Preferably, 3doses are administered, at intervals of about 3-4 months, and boostervaccinations may be given periodically thereafter. Alternate protocolsmay be appropriate for individual patients. A suitable dose is an amountof polypeptide or DNA that, when administered as described above, iscapable of raising an immune response in an immunized patient sufficientto protect the patient from M. tuberculosis infection for at least 1-2years. In general, the amount of polypeptide present in a dose (orproduced in situ by the DNA in a dose) ranges from about 1 pg to about100 mg per kg of host, typically from about 10 pg to about 1 mg, andpreferably from about 100 pg to about 1 μg. Suitable dose range willvary with the size of the patient, but will typically range from about0.1 mL to about 5 mL.

The invention having been described, the following examples are offeredby way of illustration and not limitation.

6. EXAMPLE Fusion Proteins of M. Tuberculosis Antigens RetainImmunogenicity of the Individual Components

6.1. Materials and Methods

6.1.1. Construction of Fusion Proteins

Coding sequences were modified by PCR and cloned into an expressionplasmid. Each plasmid contained three coding sequences ligated end toend. The fusion polynucleotide was inserted into vector pET-12b. Threecoding sequences for antigens Ra12, TbH9 and Ra35 were ligated to encodeone fusion protein (SEQ ID NOS:1 and 2). Another three coding sequencesfor antigens Erd14, DPV and MTI were ligated to encode a second fusionprotein (SEQ ID NOS:3 and 4). The recombinant proteins were expressed inE. coil with six histidine residues at the amino-terminal portion usingthe pET plasmid vector (pET-17b) and a T7 RNA polymerase expressionsystem (Novagen, Madison, Wis.). E. coli strain BL21 (DE3) pLysE(Novagen) was used for high level expression.

The recombinant (His-Tag) fusion proteins were purified from the solublesupernatant or the insoluble inclusion body of 500 ml of IPTG inducedbatch cultures by affinity chromatography using the one step QIAexpressNi-NTA Agarose matrix (QIAGEN, Chatsworth, Calif.) in the presence of 8Murea. Briefly, 20 ml of an overnight saturated culture of BL21containing the pET construct was added into 500 ml of 2×YT mediacontaining 50 ug/ml ampicillin and 34 ug/ml chloramphenicol, grown at37° C. with shaking. The bacterial cultures were induced with 2 mM IPTGat an OD 560 of 0.3 and grown for an additional 3 h (OD=1.3 to 1.9).Cells were harvested from 500 ml batch cultures by centrifugation andresuspended in 20 ml of binding buffer (0.1 M sodium phosphate, pH 8.0;10 mM Tris-HCl, pH 8.0) containing 2 mM PMSF and 20 ug/ml leupeptin plusone Complete protease inhibitor tablet (Boehringer Mannheim) per 25 ml.E. coli was lysed by freeze-thaw followed by brief sonication, then spunat 12 k rpm for 30 mm to pellet the inclusion bodies.

The inclusion bodies were washed three times in 1% CHAPS in 10 mMTris-HCl (pH 8.0). This step greatly reduced the level of contaminatingLPS. The inclusion body was finally solubilized in 20 ml of bindingbuffer containing 8 M urea or 8M urea was added directly into thesoluble supernatant. Recombinant fusion proteins with His-Tag residueswere batch bound to Ni-NTA agarose resin (5 ml resin per 500 mlinductions) by rocking at room temperature for 1 h and the complexpassed over a column. The flow through was passed twice over the samecolumn and the column washed three times with 30 ml each of wash buffer(0.1 M sodium phosphate and 10 mM Tris-HCL, pH 6.3) also containing 8 Murea. Bound protein was eluted with 30 ml of 150 mM immidazole in washbuffer and 5 ml fractions collected. Fractions containing eachrecombinant fusion protein were pooled, dialyzed against 10 mM TrisHCl(pH 8.0) bound one more time to the Ni-NTA matrix, eluted and dialyzedin 10 mM Tris-HCL (pH 7.8). The yield of recombinant protein varies from25-150 mg per liter of induced bacterial culture with greater than 98%purity. Recombinant proteins were assayed for endotoxin contaminationusing the Limulus assay (BioWhittaker) and were shown to contain <10E.U.Img.

6.1.2. Interferon-γ Assay

Spleens from mice were removed asceptically and single cell suspensionprepared in complete RPMI following lysis of red blood cells. 100 ul ofcells (2×10⁵ cells) were plated per well in a 96-well flat bottommicrotiter plate. Cultures were stimulated with the indicatedrecombinant proteins for 24 h and the supernatant assayed for IFN-γ.

The levels of supernatant IFN-γ was analysed by sandwich ELISA, usingantibody pairs and procedures available from PharMingen. Standard curveswere generated using recombinant mouse cytokines. ELISA plates (Coming)were coated with 50 μl/well (1 μg/ml, in 0.1 M bicarbonate coatingbuffer, pH9.6) of a cytokine capture mAb (rat anti-mouse IFN-γ(PharMingen; Cat. #181 81D)), and incubated for 4 hr at room temp. Shakeout plate contents and block with PBS-0.05% Tween, 1.0% BSA (200μl/well) overnight at 4° C. and washed for 6× in PBS-0.1% Tween.Standards (mouse IFN-γ) and supernatant samples diluted in PBS-0.05%Tween, 0.1% BSA were then added for 2 hr at room temp. The plates werewashed as above and then incubated for 2 hr at room temperature with 100ul/well of a second Ab (biotin rat α mouse IFN-γ (Cat. #18112D;PharMingen) at 0.5 μg/ml diluted in PBS-0.05% Tween, 0.1% BSA. Afterwashing, plates were incubated with 100 μl/well of streptavidin-HRP(Zymed) at a 1:2500 dilution in PBS-0.05% Tween, 0.1% BSA at room tempfor 1 hr. The plates were washed one last time and developed with 100μl/well TMB substrate (3,3′,5,5′-tetramethylbenzidine, Kirkegaard andPerry, Gaithersburg, Md.) and the reaction stopped after color develops,with H₂SO₄, 50 μl/well. Absorbance (OD) were determined at 450 nm using570 nm as a reference wavelength and the cytokine concentrationevaluated using the standard curve.

6.2. Results

Three coding sequences for M. tuberculosis antigens were inserted intoan expression vector for the production of a fusion protein. Theantigens designated Ra12, TbH9 and Ra35 were produced as one recombinantfusion protein (FIGS. 1A and B). Antigens Erd14, DPV and MTI wereproduced as a second fusion protein (FIG. 2). The two fusion proteinswere affinity purified for use in in vitro and in vivo assays.

The two fusion proteins were tested for their ability to stimulate Tcell responses from six PPD⁺ subjects. When T cell proliferation wasmeasured, both fusion proteins exhibited a similar reactivity pattern astheir individual components (FIGS. 3A-3F). A similar result was obtainedwhen IFN-γ production was measured (FIGS. 4A-4F). For example, subjectD160 responded to antigens TbH9 and MTI individually. Subject D160 alsoresponded to the fusion proteins that contained these antigens (FIGS. 3Band 4B). In contrast, no T cell response from D160 was observed to otherantigens individually. Another subject, D201, who did not react withantigens Erd14, DPV or MTI individually, was also unresponsive to thefusion protein containing these antigens. It should be noted that whenthe T cell responses to the individual components of the two fusionproteins were not particularly strong, the fusion proteins stimulatedresponses that were equal to or higher than that induced by theindividual antigens in most cases.

The Ra12-TbH9-Ra35 tri-fusion protein was also tested as an immunogen invivo. In these experiments, the fusion protein was injected into thefootpads of mice for immunization. Each group of three mice received theprotein in a different adjuvant formulation: SBAS1c, SBAS2 (Ling et al.,1997, Vaccine 15:1562-1567), SBAS7 and AL(OH)₃. After two subcutaneousimmunizations at three week intervals, the animals were sacrificed oneweek later, and their draining lymph nodes were harvested for use asresponder cells in T cell proliferation and cytokine production assays.

Regardless which adjuvant was used in the immunization, strong T cellproliferation responses were induced against TbH9 when it was used as anindividual antigen (FIG. 5A). Weaker responses were induced against Ra35and Ra12 (FIGS. 5B and 5C). When the Ra12-TbH9-Ra35 fusion protein wasused as immunogen, a response similar to that against the individualcomponents was observed.

When cytokine production was measured, adjuvants SBAS1c and SBAS2produced similar IFN-γ (FIG. 6) and IL-4 responses (FIG. 7). However,the combination of SBAS7 and aluminum hydroxide produced the strongestIFN-γ responses and the lowest level of IL-4 production for all threeantigens. With respect to the humoral antibody response in vivo, FIGS.8A-8F shows that the fusion protein elicited both IgG₁ and IgG_(2a),antigen-specific responses when it was used with any of the threeadjuvants.

Additionally, C57BL/6 mice were immunized with a combination of twoexpression constructs each containing Mtb32A or Mtb39A coding sequenceas DNA vaccines. The immunized animals exhibited significant protectionagainst tuberculosis upon a subsequent aerosol challenge of livebacteria. Based on these results, a fusion construct of Mtb32A andMtb39A coding sequences was made, and its encoded product tested in aguinea pig long term protection model. In these studies, guinea pigswere immunized with a single recombinant fusion protein or a mixture ofMtb32A and Mtb39A proteins in formulations containing an adjuvant. FIGS.9A-9C shows that guinea pigs immunized with the fusion protein in SBAS1cor SBAS2 were better protected against the development of tuberculosisupon subsequent challenge, as compared to animals immunized with the twoantigens in a mixture in the same adjuvant formulation. The fusionprotein in BAS2 formulation afforded the greatest protection in theanimals. Thus, fusion proteins of various M. tuberculosis antigens maybe used as more effective immunogens in vaccine formulations than amixture of the individual components.

The present invention is not to be limited in scope by the exemplifiedembodiments which are intended as illustrations of single aspects of theinvention, and any clones, nucleotide or amino acid sequences which arefunctionally equivalent are within the scope of the invention. Indeed,various modifications of the invention in addition to those describedherein will become apparent to those skilled in the art from theforegoing description and accompanying drawings. Such modifications areintended to fall within the scope of the appended claims. It is also tobe understood that all base pair sizes given for nucleotides areapproximate and are used for purposes of description.

All publications cited herein are incorporated by reference in theirentirety.

10 1 2287 DNA Artificial Sequence Description of ArtificialSequencetri-fusion protein Ra12-TbH9-Ra35 1 tctagaaata attttgtttactttaagaan ganatataca t atg cat cac cat cac 56 Met His His His His 1 5cat cac acg gcc gcg tcc gat aac ttc cag ctg tcc cag ggt ggg cag 104 HisHis Thr Ala Ala Ser Asp Asn Phe Gln Leu Ser Gln Gly Gly Gln 10 15 20 ggattc gcc att ccg atc ggg cag gcg atg gcg atc gcg ggc cag atc 152 Gly PheAla Ile Pro Ile Gly Gln Ala Met Ala Ile Ala Gly Gln Ile 25 30 35 cga tcgggt ggg ggg tca ccc acc gtt cat atc ggg cct acc gcc ttc 200 Arg Ser GlyGly Gly Ser Pro Thr Val His Ile Gly Pro Thr Ala Phe 40 45 50 ctc ggc ttgggt gtt gtc gac aac aac ggc aac ggc gca cga gtc caa 248 Leu Gly Leu GlyVal Val Asp Asn Asn Gly Asn Gly Ala Arg Val Gln 55 60 65 cgc gtg gtc gggagc gct ccg gcg gca agt ctc ggc atc tcc acc ggc 296 Arg Val Val Gly SerAla Pro Ala Ala Ser Leu Gly Ile Ser Thr Gly 70 75 80 85 gac gtg atc accgcg gtc gac ggc gct ccg atc aac tcg gcc acc gcg 344 Asp Val Ile Thr AlaVal Asp Gly Ala Pro Ile Asn Ser Ala Thr Ala 90 95 100 atg gcg gac gcgctt aac ggg cat cat ccc ggt gac gtc atc tcg gtg 392 Met Ala Asp Ala LeuAsn Gly His His Pro Gly Asp Val Ile Ser Val 105 110 115 acc tgg caa accaag tcg ggc ggc acg cgt aca ggg aac gtg aca ttg 440 Thr Trp Gln Thr LysSer Gly Gly Thr Arg Thr Gly Asn Val Thr Leu 120 125 130 gcc gag gga cccccg gcc gaa ttc atg gtg gat ttc ggg gcg tta cca 488 Ala Glu Gly Pro ProAla Glu Phe Met Val Asp Phe Gly Ala Leu Pro 135 140 145 ccg gag atc aactcc gcg agg atg tac gcc ggc ccg ggt tcg gcc tcg 536 Pro Glu Ile Asn SerAla Arg Met Tyr Ala Gly Pro Gly Ser Ala Ser 150 155 160 165 ctg gtg gccgcg gct cag atg tgg gac agc gtg gcg agt gac ctg ttt 584 Leu Val Ala AlaAla Gln Met Trp Asp Ser Val Ala Ser Asp Leu Phe 170 175 180 tcg gcc gcgtcg gcg ttt cag tcg gtg gtc tgg ggt ctg acg gtg ggg 632 Ser Ala Ala SerAla Phe Gln Ser Val Val Trp Gly Leu Thr Val Gly 185 190 195 tcg tgg ataggt tcg tcg gcg ggt ctg atg gtg gcg gcg gcc tcg ccg 680 Ser Trp Ile GlySer Ser Ala Gly Leu Met Val Ala Ala Ala Ser Pro 200 205 210 tat gtg gcgtgg atg agc gtc acc gcg ggg cag gcc gag ctg acc gcc 728 Tyr Val Ala TrpMet Ser Val Thr Ala Gly Gln Ala Glu Leu Thr Ala 215 220 225 gcc cag gtccgg gtt gct gcg gcg gcc tac gag acg gcg tat ggg ctg 776 Ala Gln Val ArgVal Ala Ala Ala Ala Tyr Glu Thr Ala Tyr Gly Leu 230 235 240 245 acg gtgccc ccg ccg gtg atc gcc gag aac cgt gct gaa ctg atg att 824 Thr Val ProPro Pro Val Ile Ala Glu Asn Arg Ala Glu Leu Met Ile 250 255 260 ctg atagcg acc aac ctc ttg ggg caa aac acc ccg gcg atc gcg gtc 872 Leu Ile AlaThr Asn Leu Leu Gly Gln Asn Thr Pro Ala Ile Ala Val 265 270 275 aac gaggcc gaa tac ggc gag atg tgg gcc caa gac gcc gcc gcg atg 920 Asn Glu AlaGlu Tyr Gly Glu Met Trp Ala Gln Asp Ala Ala Ala Met 280 285 290 ttt ggctac gcc gcg gcg acg gcg acg gcg acg gcg acg ttg ctg ccg 968 Phe Gly TyrAla Ala Ala Thr Ala Thr Ala Thr Ala Thr Leu Leu Pro 295 300 305 ttc gaggag gcg ccg gag atg acc agc gcg ggt ggg ctc ctc gag cag 1016 Phe Glu GluAla Pro Glu Met Thr Ser Ala Gly Gly Leu Leu Glu Gln 310 315 320 325 gccgcc gcg gtc gag gag gcc tcc gac acc gcc gcg gcg aac cag ttg 1064 Ala AlaAla Val Glu Glu Ala Ser Asp Thr Ala Ala Ala Asn Gln Leu 330 335 340 atgaac aat gtg ccc cag gcg ctg caa cag ctg gcc cag ccc acg cag 1112 Met AsnAsn Val Pro Gln Ala Leu Gln Gln Leu Ala Gln Pro Thr Gln 345 350 355 ggcacc acg cct tct tcc aag ctg ggt ggc ctg tgg aag acg gtc tcg 1160 Gly ThrThr Pro Ser Ser Lys Leu Gly Gly Leu Trp Lys Thr Val Ser 360 365 370 ccgcat cgg tcg ccg atc agc aac atg gtg tcg atg gcc aac aac cac 1208 Pro HisArg Ser Pro Ile Ser Asn Met Val Ser Met Ala Asn Asn His 375 380 385 atgtcg atg acc aac tcg ggt gtg tcg atg acc aac acc ttg agc tcg 1256 Met SerMet Thr Asn Ser Gly Val Ser Met Thr Asn Thr Leu Ser Ser 390 395 400 405atg ttg aag ggc ttt gct ccg gcg gcg gcc cgc cag gcc gtg caa acc 1304 MetLeu Lys Gly Phe Ala Pro Ala Ala Ala Arg Gln Ala Val Gln Thr 410 415 420gcg gcg caa aac ggg gtc cgg gcg atg agc tcg ctg ggc agc tcg ctg 1352 AlaAla Gln Asn Gly Val Arg Ala Met Ser Ser Leu Gly Ser Ser Leu 425 430 435ggt tct tcg ggt ctg ggc ggt ggg gtg gcc gcc aac ttg ggt cgg gcg 1400 GlySer Ser Gly Leu Gly Gly Gly Val Ala Ala Asn Leu Gly Arg Ala 440 445 450gcc tcg gtc ggt tcg ttg tcg gtg ccg cag gcc tgg gcc gcg gcc aac 1448 AlaSer Val Gly Ser Leu Ser Val Pro Gln Ala Trp Ala Ala Ala Asn 455 460 465cag gca gtc acc ccg gcg gcg cgg gcg ctg ccg ctg acc agc ctg acc 1496 GlnAla Val Thr Pro Ala Ala Arg Ala Leu Pro Leu Thr Ser Leu Thr 470 475 480485 agc gcc gcg gaa aga ggg ccc ggg cag atg ctg ggc ggg ctg ccg gtg 1544Ser Ala Ala Glu Arg Gly Pro Gly Gln Met Leu Gly Gly Leu Pro Val 490 495500 ggg cag atg ggc gcc agg gcc ggt ggt ggg ctc agt ggt gtg ctg cgt 1592Gly Gln Met Gly Ala Arg Ala Gly Gly Gly Leu Ser Gly Val Leu Arg 505 510515 gtt ccg ccg cga ccc tat gtg atg ccg cat tct ccg gca gcc ggc gat 1640Val Pro Pro Arg Pro Tyr Val Met Pro His Ser Pro Ala Ala Gly Asp 520 525530 atc gcc ccg ccg gcc ttg tcg cag gac cgg ttc gcc gac ttc ccc gcg 1688Ile Ala Pro Pro Ala Leu Ser Gln Asp Arg Phe Ala Asp Phe Pro Ala 535 540545 ctg ccc ctc gac ccg tcc gcg atg gtc gcc caa gtg ggg cca cag gtg 1736Leu Pro Leu Asp Pro Ser Ala Met Val Ala Gln Val Gly Pro Gln Val 550 555560 565 gtc aac atc aac acc aaa ctg ggc tac aac aac gcc gtg ggc gcc ggg1784 Val Asn Ile Asn Thr Lys Leu Gly Tyr Asn Asn Ala Val Gly Ala Gly 570575 580 acc ggc atc gtc atc gat ccc aac ggt gtc gtg ctg acc aac aac cac1832 Thr Gly Ile Val Ile Asp Pro Asn Gly Val Val Leu Thr Asn Asn His 585590 595 gtg atc gcg ggc gcc acc gac atc aat gcg ttc agc gtc ggc tcc ggc1880 Val Ile Ala Gly Ala Thr Asp Ile Asn Ala Phe Ser Val Gly Ser Gly 600605 610 caa acc tac ggc gtc gat gtg gtc ggg tat gac cgc acc cag gat gtc1928 Gln Thr Tyr Gly Val Asp Val Val Gly Tyr Asp Arg Thr Gln Asp Val 615620 625 gcg gtg ctg cag ctg cgc ggt gcc ggt ggc ctg ccg tcg gcg gcg atc1976 Ala Val Leu Gln Leu Arg Gly Ala Gly Gly Leu Pro Ser Ala Ala Ile 630635 640 645 ggt ggc ggc gtc gcg gtt ggt gag ccc gtc gtc gcg atg ggc aacagc 2024 Gly Gly Gly Val Ala Val Gly Glu Pro Val Val Ala Met Gly Asn Ser650 655 660 ggt ggg cag ggc gga acg ccc cgt gcg gtg cct ggc agg gtg gtcgcg 2072 Gly Gly Gln Gly Gly Thr Pro Arg Ala Val Pro Gly Arg Val Val Ala665 670 675 ctc ggc caa acc gtg cag gcg tcg gat tcg ctg acc ggt gcc gaagag 2120 Leu Gly Gln Thr Val Gln Ala Ser Asp Ser Leu Thr Gly Ala Glu Glu680 685 690 aca ttg aac ggg ttg atc cag ttc gat gcc gcg atc cag ccc ggtgat 2168 Thr Leu Asn Gly Leu Ile Gln Phe Asp Ala Ala Ile Gln Pro Gly Asp695 700 705 tcg ggc ggg ccc gtc gtc aac ggc cta gga cag gtg gtc ggt atgaac 2216 Ser Gly Gly Pro Val Val Asn Gly Leu Gly Gln Val Val Gly Met Asn710 715 720 725 acg gcc gcg tcc taggatatcc atcacactgg cggccgctcgagcagatccg 2268 Thr Ala Ala Ser gntgtaacaa agcccgaaa 2287 2 729 PRTArtificial Sequence Description of Artificial Sequencetri-fusion 2 MetHis His His His His His Thr Ala Ala Ser Asp Asn Phe Gln Leu 1 5 10 15Ser Gln Gly Gly Gln Gly Phe Ala Ile Pro Ile Gly Gln Ala Met Ala 20 25 30Ile Ala Gly Gln Ile Arg Ser Gly Gly Gly Ser Pro Thr Val His Ile 35 40 45Gly Pro Thr Ala Phe Leu Gly Leu Gly Val Val Asp Asn Asn Gly Asn 50 55 60Gly Ala Arg Val Gln Arg Val Val Gly Ser Ala Pro Ala Ala Ser Leu 65 70 7580 Gly Ile Ser Thr Gly Asp Val Ile Thr Ala Val Asp Gly Ala Pro Ile 85 9095 Asn Ser Ala Thr Ala Met Ala Asp Ala Leu Asn Gly His His Pro Gly 100105 110 Asp Val Ile Ser Val Thr Trp Gln Thr Lys Ser Gly Gly Thr Arg Thr115 120 125 Gly Asn Val Thr Leu Ala Glu Gly Pro Pro Ala Glu Phe Met ValAsp 130 135 140 Phe Gly Ala Leu Pro Pro Glu Ile Asn Ser Ala Arg Met TyrAla Gly 145 150 155 160 Pro Gly Ser Ala Ser Leu Val Ala Ala Ala Gln MetTrp Asp Ser Val 165 170 175 Ala Ser Asp Leu Phe Ser Ala Ala Ser Ala PheGln Ser Val Val Trp 180 185 190 Gly Leu Thr Val Gly Ser Trp Ile Gly SerSer Ala Gly Leu Met Val 195 200 205 Ala Ala Ala Ser Pro Tyr Val Ala TrpMet Ser Val Thr Ala Gly Gln 210 215 220 Ala Glu Leu Thr Ala Ala Gln ValArg Val Ala Ala Ala Ala Tyr Glu 225 230 235 240 Thr Ala Tyr Gly Leu ThrVal Pro Pro Pro Val Ile Ala Glu Asn Arg 245 250 255 Ala Glu Leu Met IleLeu Ile Ala Thr Asn Leu Leu Gly Gln Asn Thr 260 265 270 Pro Ala Ile AlaVal Asn Glu Ala Glu Tyr Gly Glu Met Trp Ala Gln 275 280 285 Asp Ala AlaAla Met Phe Gly Tyr Ala Ala Ala Thr Ala Thr Ala Thr 290 295 300 Ala ThrLeu Leu Pro Phe Glu Glu Ala Pro Glu Met Thr Ser Ala Gly 305 310 315 320Gly Leu Leu Glu Gln Ala Ala Ala Val Glu Glu Ala Ser Asp Thr Ala 325 330335 Ala Ala Asn Gln Leu Met Asn Asn Val Pro Gln Ala Leu Gln Gln Leu 340345 350 Ala Gln Pro Thr Gln Gly Thr Thr Pro Ser Ser Lys Leu Gly Gly Leu355 360 365 Trp Lys Thr Val Ser Pro His Arg Ser Pro Ile Ser Asn Met ValSer 370 375 380 Met Ala Asn Asn His Met Ser Met Thr Asn Ser Gly Val SerMet Thr 385 390 395 400 Asn Thr Leu Ser Ser Met Leu Lys Gly Phe Ala ProAla Ala Ala Arg 405 410 415 Gln Ala Val Gln Thr Ala Ala Gln Asn Gly ValArg Ala Met Ser Ser 420 425 430 Leu Gly Ser Ser Leu Gly Ser Ser Gly LeuGly Gly Gly Val Ala Ala 435 440 445 Asn Leu Gly Arg Ala Ala Ser Val GlySer Leu Ser Val Pro Gln Ala 450 455 460 Trp Ala Ala Ala Asn Gln Ala ValThr Pro Ala Ala Arg Ala Leu Pro 465 470 475 480 Leu Thr Ser Leu Thr SerAla Ala Glu Arg Gly Pro Gly Gln Met Leu 485 490 495 Gly Gly Leu Pro ValGly Gln Met Gly Ala Arg Ala Gly Gly Gly Leu 500 505 510 Ser Gly Val LeuArg Val Pro Pro Arg Pro Tyr Val Met Pro His Ser 515 520 525 Pro Ala AlaGly Asp Ile Ala Pro Pro Ala Leu Ser Gln Asp Arg Phe 530 535 540 Ala AspPhe Pro Ala Leu Pro Leu Asp Pro Ser Ala Met Val Ala Gln 545 550 555 560Val Gly Pro Gln Val Val Asn Ile Asn Thr Lys Leu Gly Tyr Asn Asn 565 570575 Ala Val Gly Ala Gly Thr Gly Ile Val Ile Asp Pro Asn Gly Val Val 580585 590 Leu Thr Asn Asn His Val Ile Ala Gly Ala Thr Asp Ile Asn Ala Phe595 600 605 Ser Val Gly Ser Gly Gln Thr Tyr Gly Val Asp Val Val Gly TyrAsp 610 615 620 Arg Thr Gln Asp Val Ala Val Leu Gln Leu Arg Gly Ala GlyGly Leu 625 630 635 640 Pro Ser Ala Ala Ile Gly Gly Gly Val Ala Val GlyGlu Pro Val Val 645 650 655 Ala Met Gly Asn Ser Gly Gly Gln Gly Gly ThrPro Arg Ala Val Pro 660 665 670 Gly Arg Val Val Ala Leu Gly Gln Thr ValGln Ala Ser Asp Ser Leu 675 680 685 Thr Gly Ala Glu Glu Thr Leu Asn GlyLeu Ile Gln Phe Asp Ala Ala 690 695 700 Ile Gln Pro Gly Asp Ser Gly GlyPro Val Val Asn Gly Leu Gly Gln 705 710 715 720 Val Val Gly Met Asn ThrAla Ala Ser 725 3 1081 DNA Artificial Sequence Description of ArtificialSequencetri-fusion protein Erd14-DPV-MTI 3 gatatacat atg cat cac cat caccat cac atg gcc acc acc ctt ccc gtt 51 Met His His His His His His MetAla Thr Thr Leu Pro Val 1 5 10 cag cgc cac ccg cgg tcc ctc ttc ccc gagttt tct gag ctg ttc gcg 99 Gln Arg His Pro Arg Ser Leu Phe Pro Glu PheSer Glu Leu Phe Ala 15 20 25 30 gcc ttc ccg tca ttc gcc gga ctc cgg cccacc ttc gac acc cgg ttg 147 Ala Phe Pro Ser Phe Ala Gly Leu Arg Pro ThrPhe Asp Thr Arg Leu 35 40 45 atg cgg ctg gaa gac gag atg aaa gag ggg cgctac gag gta cgc gcg 195 Met Arg Leu Glu Asp Glu Met Lys Glu Gly Arg TyrGlu Val Arg Ala 50 55 60 gag ctt ccc ggg gtc gac ccc gac aag gac gtc gacatt atg gtc cgc 243 Glu Leu Pro Gly Val Asp Pro Asp Lys Asp Val Asp IleMet Val Arg 65 70 75 gat ggt cag ctg acc atc aag gcc gag cgc acc gag cagaag gac ttc 291 Asp Gly Gln Leu Thr Ile Lys Ala Glu Arg Thr Glu Gln LysAsp Phe 80 85 90 gac ggt cgc tcg gaa ttc gcg tac ggt tcc ttc gtt cgc acggtg tcg 339 Asp Gly Arg Ser Glu Phe Ala Tyr Gly Ser Phe Val Arg Thr ValSer 95 100 105 110 ctg ccg gta ggt gct gac gag gac gac att aag gcc acctac gac aag 387 Leu Pro Val Gly Ala Asp Glu Asp Asp Ile Lys Ala Thr TyrAsp Lys 115 120 125 ggc att ctt act gtg tcg gtg gcg gtt tcg gaa ggg aagcca acc gaa 435 Gly Ile Leu Thr Val Ser Val Ala Val Ser Glu Gly Lys ProThr Glu 130 135 140 aag cac att cag atc cgg tcc acc aac aag ctt gat cccgtg gac gcg 483 Lys His Ile Gln Ile Arg Ser Thr Asn Lys Leu Asp Pro ValAsp Ala 145 150 155 gtc att aac acc acc tgc aat tac ggg cag gta gta gctgcg ctc aac 531 Val Ile Asn Thr Thr Cys Asn Tyr Gly Gln Val Val Ala AlaLeu Asn 160 165 170 gcg acg gat ccg ggg gct gcc gca cag ttc aac gcc tcaccg gtg gcg 579 Ala Thr Asp Pro Gly Ala Ala Ala Gln Phe Asn Ala Ser ProVal Ala 175 180 185 190 cag tcc tat ttg cgc aat ttc ctc gcc gca ccg ccacct cag cgc gct 627 Gln Ser Tyr Leu Arg Asn Phe Leu Ala Ala Pro Pro ProGln Arg Ala 195 200 205 gcc atg gcc gcg caa ttg caa gct gtg ccg ggg gcggca cag tac atc 675 Ala Met Ala Ala Gln Leu Gln Ala Val Pro Gly Ala AlaGln Tyr Ile 210 215 220 ggc ctt gtc gag tcg gtt gcc ggc tcc tgc aac aactat gag ctc atg 723 Gly Leu Val Glu Ser Val Ala Gly Ser Cys Asn Asn TyrGlu Leu Met 225 230 235 acg att aat tac cag ttc ggg gac gtc gac gct catggc gcc atg atc 771 Thr Ile Asn Tyr Gln Phe Gly Asp Val Asp Ala His GlyAla Met Ile 240 245 250 cgc gct cag gcg gcg tcg ctt gag gcg gag cat caggcc atc gtt cgt 819 Arg Ala Gln Ala Ala Ser Leu Glu Ala Glu His Gln AlaIle Val Arg 255 260 265 270 gat gtg ttg gcc gcg ggt gac ttt tgg ggc ggcgcc ggt tcg gtg gct 867 Asp Val Leu Ala Ala Gly Asp Phe Trp Gly Gly AlaGly Ser Val Ala 275 280 285 tgc cag gag ttc att acc cag ttg ggc cgt aacttc cag gtg atc tac 915 Cys Gln Glu Phe Ile Thr Gln Leu Gly Arg Asn PheGln Val Ile Tyr 290 295 300 gag cag gcc aac gcc cac ggg cag aag gtg caggct gcc ggc aac aac 963 Glu Gln Ala Asn Ala His Gly Gln Lys Val Gln AlaAla Gly Asn Asn 305 310 315 atg gcg caa acc gac agc gcc gtc ggc tcc agctgg gcc actagtaacg 1012 Met Ala Gln Thr Asp Ser Ala Val Gly Ser Ser TrpAla 320 325 330 gccgccagtg tgctggaatt ctgcagatat ccatcacact ggcggccgctcgagcagatc 1072 cggctgcta 1081 4 331 PRT Artificial Sequence Descriptionof Artificial Sequencetri-fusion 4 Met His His His His His His Met AlaThr Thr Leu Pro Val Gln Arg 1 5 10 15 His Pro Arg Ser Leu Phe Pro GluPhe Ser Glu Leu Phe Ala Ala Phe 20 25 30 Pro Ser Phe Ala Gly Leu Arg ProThr Phe Asp Thr Arg Leu Met Arg 35 40 45 Leu Glu Asp Glu Met Lys Glu GlyArg Tyr Glu Val Arg Ala Glu Leu 50 55 60 Pro Gly Val Asp Pro Asp Lys AspVal Asp Ile Met Val Arg Asp Gly 65 70 75 80 Gln Leu Thr Ile Lys Ala GluArg Thr Glu Gln Lys Asp Phe Asp Gly 85 90 95 Arg Ser Glu Phe Ala Tyr GlySer Phe Val Arg Thr Val Ser Leu Pro 100 105 110 Val Gly Ala Asp Glu AspAsp Ile Lys Ala Thr Tyr Asp Lys Gly Ile 115 120 125 Leu Thr Val Ser ValAla Val Ser Glu Gly Lys Pro Thr Glu Lys His 130 135 140 Ile Gln Ile ArgSer Thr Asn Lys Leu Asp Pro Val Asp Ala Val Ile 145 150 155 160 Asn ThrThr Cys Asn Tyr Gly Gln Val Val Ala Ala Leu Asn Ala Thr 165 170 175 AspPro Gly Ala Ala Ala Gln Phe Asn Ala Ser Pro Val Ala Gln Ser 180 185 190Tyr Leu Arg Asn Phe Leu Ala Ala Pro Pro Pro Gln Arg Ala Ala Met 195 200205 Ala Ala Gln Leu Gln Ala Val Pro Gly Ala Ala Gln Tyr Ile Gly Leu 210215 220 Val Glu Ser Val Ala Gly Ser Cys Asn Asn Tyr Glu Leu Met Thr Ile225 230 235 240 Asn Tyr Gln Phe Gly Asp Val Asp Ala His Gly Ala Met IleArg Ala 245 250 255 Gln Ala Ala Ser Leu Glu Ala Glu His Gln Ala Ile ValArg Asp Val 260 265 270 Leu Ala Ala Gly Asp Phe Trp Gly Gly Ala Gly SerVal Ala Cys Gln 275 280 285 Glu Phe Ile Thr Gln Leu Gly Arg Asn Phe GlnVal Ile Tyr Glu Gln 290 295 300 Ala Asn Ala His Gly Gln Lys Val Gln AlaAla Gly Asn Asn Met Ala 305 310 315 320 Gln Thr Asp Ser Ala Val Gly SerSer Trp Ala 325 330 5 3 PRT Artificial Sequence Description ofArtificial Sequenceflexible polylinker 5 Gly Cys Gly 1 6 6 PRTArtificial Sequence Description of Artificial Sequenceflexiblepolylinker 6 Gly Cys Gly Gly Cys Gly 1 5 7 9 PRT Artificial SequenceDescription of Artificial Sequenceflexible polylinker 7 Gly Cys Gly GlyCys Gly Gly Cys Gly 1 5 8 5 PRT Artificial Sequence Description ofArtificial Sequenceflexible polylinker 8 Gly Gly Gly Gly Ser 1 5 9 10PRT Artificial Sequence Description of Artificial Sequenceflexiblepolylinker 9 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 1 5 10 10 15 PRTArtificial Sequence Description of Artificial Sequenceflexiblepolylinker 10 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly GlySer 1 5 10 15

What is claimed is:
 1. A purified polypeptide comprising the amino acid sequence of SEQ ID NO:
 2. 2. The polypeptide of claim 1 which is a soluble polypeptide.
 3. The polypeptide of claim 1 which is produced by a recombinant DNA method.
 4. The polypeptide of claim 1 which is produced by a chemical synthetic method.
 5. The polypeptide of claim 1 which is fused with a second heterologous polypeptide.
 6. A pharmaceutical composition comprising the polypeptide of claim
 1. 7. The composition of claim 6, further comprising an adjuvant.
 8. The composition of claim 6, wherein the composition is formulated in an oil emulsion. 